The proportion of cells in the DNA synthesis phase provides a reliable indication of the proliferative capacity of a cell population. Click-iT® EdU can be used to evaluate proliferation dynamics in primary human cells (an important experimental model). As shown in Figure 1, for Gibco® Human Skeletal Myoblasts (HSkM) plated at three different densities and grown in differentiation media for 48 hours, higher plating densities correlated with a lower proliferative fraction of cells.

Click-iT® EdU can also be used to detect variations in the proliferative fraction of cells due to cell cycle disruption. Figure 2 shows how Click-iT® EdU assays were used with several cultured cell lines to directly measure the percentage of cells in S phase. Dual-parameter analysis of DNA content vs. Click-iT® EdU fluorescence clearly identifies cells in different phases of the cell cycle. The percentage of cells in S phase provides an indication of the degree of cell proliferation.

Figure 2. Dual-parameter analysis of cell cycle perturbation. Gibco® Primary Adult Human Dermal Fibroblasts (HDFa), cervical carcinoma cells (HeLa), and human alveolar epithelial cells (A549) were grown in culture for 48 hr. Cells were either treated with a 500 nM pulse of the antimitotic drug Paclitaxel or left untreated. Cells were treated with 10 µM EdU for 2 hr prior to analysis. Incorporated EdU was detected using a reaction with Click-iT® EdU Alexa Fluor® 488 dye azide. Cells were analyzed using the Attune® Acoustic Focusing Cytometer with 405 nm and 488 nm lasers using standard VL1 (450/40) and BL1 (530/30) emission filters. Dual-parameter density plots of FxCycle™ Violet vs. Alexa Fluor® 488 Click-iT® EdU fluorescence clearly identified cells in the G0/G1, S, and G2/M phases of the cell cycle. Untreated A549 cells had a significantly higher percentage of cells in S phase and a lower percentage in G0/G1 than other cell types, reflecting a higher proliferative index. Upon treatment with paclitaxel, more than 90% of A549 and HeLa cells were arrested in G2 phase due to mitotic inhibition. A similar pattern of arrest was seen with HDFa, with the exception of approximately 26% of cells which remained in the quiescent G0 state.

An Easier Way to Analyze Cell Proliferation

The unique chemistry of assays based on Click-iT® EdU technology enables a simple and efficient method for evaluating cell proliferation. These new assays help researchers to accurately correlate proliferative dynamics with different plating densities, and clearly identify cells in the S phase of the cell cycle. Click-iT® EdU labeling is compatible with most fixation protocols.

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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.